Chemo-enzymatic site-specific modification of peptides and proteins to form cleavable conjugates

Abstract

A method is provided for reversibly modifying a protein or peptide on its glutamine residue(s) by performing a reaction, such as a transglutaminase-catalyzed reaction, between the protein or peptide and an amine-containing reagent, whereby the reagent is linked through its amine function to a side chain of the glutamine residue. Subjecting the modified protein to an appropriate stimulus regenerates the protein or peptide in its original form.

What is claimed is: 
     
       1. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises a photolysis reaction; and 
 wherein the amine-containing reagent is a compound having the formula 
 
       
         
           
           
               
               
           
         
         wherein 
         Y is selected from the group consisting of hydrogen, halogen, hydroxy, thiol, cyano, isocyano, thiocyano, isothiocyano, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, (C 1 -C 3 )alkyloxy; halo(C 1 -C 3 )alkyloxy, and cyclopropyl; and 
         Z is selected from the group consisting of hydrogen; halogen; hydroxy; nitro; cyano; isocyano; thiocyano; isothiocyano; (C 1 -C 6 )alkyl; halo(C 1 -C 6 )alkyl; phenyl(C 1 -C 6 )alkyl; phenyl optionally substituted with halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl or (C 1 -C 3 )alkyloxy; (C 1 -C 3 )alkyloxy; cyclopropyl; halo(C 1 -C 3 )alkyloxy; (C 2 -C 4 )alkenyl; (C 2 -C 4 )alkynyl; (C 1 -C 6 )alkylthio; C(O)OR 1 ; COR 1 ; CON(R 1 ) 2 ; azide; sulfinyl; sulfonyl; sulfonyl halide; sulfino; sulfo; thiol; (C 4 -C 6 )diene; and aryl or aryloxy optionally substituted halogen or (C 1 -C 3 )alkyl; wherein R 1  is hydrogen, halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, or aryl optionally substituted with halogen or (C 1 -C 3 )alkyl. 
       
     
     
       2. The method of  claim 1 , further comprising carrying out said treatment of the first derivative. 
     
     
       3. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises a photolysis reaction; 
 wherein the amine-containing reagent comprises a hydroxylamine group, and the reagent is linked through the hydroxylamine group to the side chain of the at least one glutamine residue; 
 wherein the reagent comprising a hydroxylamine group is a compound having the formula 
 
       
         
           
           
               
               
           
         
         wherein 
         Y is selected from the group consisting of hydrogen, halogen, hydroxy, thiol, cyano, isocyano, thiocyano, isothiocyano, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, (C 1 -C 3 )alkyloxy; halo(C 1 -C 3 )alkyloxy, and cyclopropyl; and 
         Z is selected from the group consisting of hydrogen; halogen; hydroxy; nitro; cyano; isocyano; thiocyano; isothiocyano; (C 1 -C 6 )alkyl; halo(C 1 -C 6 )alkyl; phenyl(C 1 -C 6 )alkyl; phenyl optionally substituted with halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl or (C 1 -C 3 )alkyloxy; (C 1 -C 3 )alkyloxy; cyclopropyl; halo(C 1 -C 3 )alkyloxy; (C 2 -C 4 )alkenyl; (C 2 -C 4 )alkynyl; (C 1 -C 6 )alkylthio; C(O)OR 1 ; COR 1 ; CON(R 1 ) 2 ; azide; sulfinyl; sulfonyl; sulfonyl halide; sulfino; sulfo; thiol; (C 4 -C 6 )diene; and aryl or aryloxy optionally substituted halogen or (C 1 -C 3 )alkyl; wherein R 1  is hydrogen, halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, or aryl optionally substituted with halogen or (C 1 -C 3 )alkyl. 
       
     
     
       4. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises a photolysis reaction; 
 wherein the amine-containing reagent comprises a hydrazine group, and the reagent is linked through the hydrazine group to the side chain of the at least one glutamine residue; 
 wherein the reagent comprising a hydrazine group is a compound having the formula 
 
       
         
           
           
               
               
           
         
         wherein 
         Y, R 1  and R 2  are selected from the group consisting of hydrogen, halogen, hydroxy, thiol, cyano, isocyano, thiocyano, isothiocyano, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, (C 1 -C 3 )alkyloxy; halo(C 1 -C 3 )alkyloxy, and cyclopropyl; and 
         Z is selected from the group consisting of hydrogen; halogen; hydroxy; nitro; cyano; 
         isocyano; thiocyano; isothiocyano; (C 1 -C 6 )alkyl; halo(C 1 -C 6 )alkyl; phenyl(C 1 -C 6 )alkyl; phenyl optionally substituted with halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl or (C 1 -C 3 )alkyloxy; (C 1 -C 3 )alkyloxy; cyclopropyl; halo(C 1 -C 3 )alkyloxy; (C 2 -C 4 )alkenyl; (C 2 -C 4 )alkynyl; (C 1 -C 6 )alkylthio; C(O)OR 1 ; COR 1 ; CON(R 1 ) 2 ; azide; sulfinyl; sulfonyl; sulfonyl halide; sulfino; sulfo; thiol; (C 4 -C 6 )diene; and aryl or aryloxy optionally substituted halogen or (C 1 -C 3 )alkyl; wherein R 1  is hydrogen, halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, or aryl optionally substituted with halogen or (C 1 -C 3 )alkyl. 
       
     
     
       5. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises a reaction catalyzed by a metal in elemental form, a metal in ionic form, or a metal that is part of a complex; 
 wherein the amine-containing reagent is a reagent comprising a hydroxylamine group, wherein the reagent is linked through the hydroxylamine group to a side chain of the at least one glutamine residue; or a reagent comprising a hydrazine group, wherein the reagent is linked through the hydrazine group to a side chain of the at least one glutamine residue. 
 
     
     
       6. The method of  claim 5 , wherein the reagent comprising a hydroxylamine group is a compound having the formula 
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , and R 5  are independently selected from the group consisting of: selected from the group consisting of: hydrogen; halogen; hydroxy; nitro; cyano; isocyano; thiocyano; isothiocyano; (C 1 -C 6 )alkyl; halo(C 1 -C 6 )alkyl; phenyl(C 1 -C 6 )alkyl; phenyl optionally substituted with halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl or (C 1 -C 3 )alkyloxy; (C 1 -C 3 )alkyloxy; cyclopropyl; halo(C 1 -C 3 )alkyloxy; (C 2 -C 4 )alkenyl; (C 2 -C 4 )alkynyl; (C 1 -C 6 )alkylthio; C(O)OR 1 ; COR 1 ; CON(R 1 ) 2 ; azide; sulfinyl; sulfonyl; sulfonyl halide; sulfino; sulfo; thiol; (C 4 -C 6 )diene; and aryl or aryloxy optionally substituted halogen or (C 1 -C 3 )alkyl; wherein R 1  is hydrogen, halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, or aryl optionally substituted with halogen or (C 1 -C 3 )alkyl. 
     
     
       7. The method of  claim 5 , wherein the reagent comprising a hydrazine group is a compound having the formula 
       
         
           
           
               
               
           
         
       
       wherein
 R 1 , R 2 , R 3 , R 4 , and R 5  are independently selected from the group consisting of: selected from the group consisting of hydrogen; halogen; hydroxy; nitro; cyano; isocyano; thiocyano; isothiocyano; (C 1 -C 6 )alkyl; halo(C 1 -C 6 )alkyl; phenyl(C 1 -C 6 )alkyl; phenyl optionally substituted with halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl or (C 1 -C 3 )alkyloxy; (C 1 -C 3 )alkyloxy; cyclopropyl; halo(C 1 -C 3 )alkyloxy; (C 2 -C 4 )alkenyl; (C 2 -C 4 )alkynyl; (C 1 -C 6 )alkylthio; C(O)OR 1 ; COR 1 ; CON(R 1 ) 2 ; azide; sulfinyl; sulfonyl; sulfonyl halide; sulfino; sulfo; thiol; (C 4 -C 6 )diene; and aryl or aryloxy optionally substituted halogen or (C 1 -C 3 )alkyl; wherein is hydrogen, halogen, (C 1 -C 3 )alkyl, halo(C 1 -C 3 )alkyl, or aryl optionally substituted with halogen or (C 1 -C 3 )alkyl. 
 
     
     
       8. The method of  claim 5 , wherein the metal is palladium, iron, ruthenium, or platinum. 
     
     
       9. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein
 X is oxygen, sulfur, nitrogen, phosphorous, or selenium; 
 Y is hydrogen, alkyl, acyl, sulfo, or phospho; 
 J, K, L, and M are each independently carbon or nitrogen; 
 R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, hydroxyl, nitro, cyano, isocyano, thiocyano, isothiocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 5  is hydrogen, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       10. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 Z is alkyl, aryl, acyl, acyloxy, alkenyl, alkynyl, silyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, acyl, cyano, alkoxy, or nitroso; 
 X is oxygen, sulfur, nitrogen, phosphorous, or selenium; 
 Y is hydrogen, alkyl, dialkyl, acyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, hydroxyl, silyl, nitrosyl, thiol, sulfide, or oxo; 
 J, K, L, and M are each independently carbon or nitrogen; 
 R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, hydroxyl, nitro, nitroso, cyano, isocyano, thiocyano, isothiocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 5  is hydrogen, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       11. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 X is oxygen, sulfur, nitrogen, phosphorous, or selenium; 
 Y is hydrogen, alkyl, dialkyl, acyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, hydroxyl, silyl, nitrosyl, thiol, sulfide, or oxo; 
 J, K, L, and M are each independently carbon or nitrogen; 
 R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, hydroxyl, nitro, nitroso, cyano, isocyano, thiocyano, isothiocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 5  is hydrogen, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       12. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 X is oxygen, sulfur, nitrogen, phosphorous, or selenium; 
 Y is hydrogen, alkyl, acyl, sulfo, or phospho; 
 J, K, L, and M are each independently carbon or nitrogen; 
 R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, hydroxyl, nitro, cyano, isocyano, thiocyano, isothiocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 5  is hydrogen, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       13. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein
 Z is alkyl, aryl, acyl, acyloxy, alkenyl, alkynyl, silyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, acyl, cyano, alkoxy, or nitroso; 
 X is oxygen, sulfur, nitrogen, phosphorous, or selenium; 
 Y is hydrogen, alkyl, dialkyl, acyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, hydroxyl, silyl, nitrosyl, thiol, sulfide, or oxo; 
 J, K, L, and M are each independently carbon or nitrogen; 
 R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, hydroxyl, nitro, nitroso, cyano, isocyano, thiocyano, isothiocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 5  is hydrogen, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       14. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 X is oxygen, sulfur, nitrogen, phosphorous, or selenium; 
 Y is hydrogen, alkyl, dialkyl, acyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, hydroxyl, silyl, nitrosyl, thiol, sulfide, or oxo; 
 J, K, L, and M are each independently carbon or nitrogen; 
 R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, hydroxyl, nitro, nitroso, cyano, isocyano, thiocyano, isothiocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, thiol; and 
 R 5  is hydrogen, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       15. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 X is halogen, sulfonyl, sulfinyl, sulfoxo, cyano, isocyano, alkoxy, alkylthio, nitro, phospho, sulfo, or thiol; 
 R 1 , and R 2  are each independently hydrogen, halogen, hydroxyl, nitro, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 3  is hydrogen, cyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       16. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 Z is alkyl, aryl, acyl, acyloxy, alkenyl, alkynyl, silyl, sulfo, sulfonyl, sulfinyl, phospho, phosphonyl, acyl, cyano, alkoxy, or nitroso; 
 X is halogen, sulfonyl, sulfinyl, sulfo, sulfino, selenide, selenoxide, selenone, cyano, isocyano, alkoxy, alkylthio, nitro, amine oxide, phospho, or thiol; 
 R 1 , and R 2  are each independently hydrogen, halogen, hydroxyl, nitro, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 3  is hydrogen, cyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       17. A method for modifying a protein or peptide having one or more glutamine residues, the method comprising:
 performing a transglutaminase-catalyzed reaction between the protein or peptide and an amine-containing reagent, thereby producing a first derivative of the protein or peptide, wherein the reagent becomes covalently linked through its amine function to a side chain of at least one of said one or more glutamine residues; wherein the original protein or peptide, or a second derivative thereof, can be obtained by a treatment of the first derivative of the protein or peptide; 
 wherein the treatment comprises exposure to a physiological condition, altered pH, altered ionic strength, or elevated temperature; 
 wherein the amine-containing reagent is 
 
       
         
           
           
               
               
           
         
       
       wherein:
 X is halogen, sulfonyl, sulfinyl, sulfo, sulfino, selenide, selenoxide, selenone, cyano, isocyano, alkoxy, alkylthio, nitro, amine oxide, phospho, or thiol; 
 R 1  and R 2  are each independently hydrogen, halogen, hydroxyl, nitro, cyano, isocyano, aryl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, acyl, carbonyl, carboxyl, azide, sulfinyl, sulfonyl, sulfino, sulfo, or thiol; and 
 R 3  is hydrogen, cyano, aryl, alkyl, alkenyl, alkynyl, acyl, carbonyl, or carboxyl. 
 
     
     
       18. The method of  claim 1 , wherein a  15 N label is introduced into or removed from said one or more glutamine residues. 
     
     
       19. The method of  claim 1 , wherein the treatment comprises photolysis of the first derivative. 
     
     
       20. The method of  claim 1 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       21. The method of  claim 3 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       22. The method of  claim 4 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       23. The method of  claim 6 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       24. The method of  claim 7 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       25. The method of  claim 9 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       26. The method of  claim 10 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       27. The method of  claim 11 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       28. The method of  claim 12 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       29. The method of  claim 13 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       30. The method of  claim 14 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       31. The method of  claim 15 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       32. The method of  claim 16 , wherein a protein is modified, and wherein the protein is an enzyme. 
     
     
       33. The method of  claim 17 , wherein a protein is modified, and wherein the protein is an enzyme.